Insight into the Structural Dynamics of the Lysenin During Prepore-to-Pore Transition Using Hydrogen–Deuterium Exchange Mass Spectrometry

Abstract

Lysenin is a pore-forming toxin of the aerolysin family, which is derived from coelomic fluid of the earthworm Eisenia fetida. Upon binding to sphingomyelin (SM)-containing membranes, lysenin undergoes a series of structural changes promoting the conversion of water-soluble monomers into oligomers, leading to its insertion into the membrane and the formation of a lytic β-barrel pore. The soluble monomer and transmembrane pore structures were recently described, but the underlying structural details of oligomerization remain unclear. To investigate the molecular mechanisms controlling the conformational rearrangements accompanying pore formation, we compared the hydrogen–deuterium exchange pattern between lyseninWT and its mutant lyseninV88C/Y131C. This mutation arrests lysenin oligomers in the prepore state at the membrane surface and does not affect the structural dynamics of the water-soluble form of lysenin. In contrast, membrane-bound lyseninV88C/Y131C exhibited increased structural stabilization, especially within the twisted β-sheet of the N-terminal domain. We demonstrated that the structural stabilization of the lysenin prepore started at the site of lysenin’s initial interaction with the lipid membrane and was transmitted to the twisted β-sheet of the N-terminal domain, and that lyseninV88C/Y131C was arrested in this conformation. In lyseninWT, stabilization of these regions drove the conformational changes necessary for pore formation.

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“…Thanks to all the developments in methodology, HDX-MS has facilitated more meaningful investigations into many membrane spanning systems including, to name a few, transporters, ,− ,− ion channels, , apoptotic protein assembly, viral glycoproteins ,,,, and matrix proteins, , pore forming toxins, ATP synthase, receptor complexes, ,,, and enzymes . HDX-MS has also been used to probe peripheral membrane proteins in the context of their native environments such as drug metabolizing enzymes at the lipid membrane surface − and the interactions and structural transitions mediating lipoprotein assembly. ,,, One interesting finding from the slew of HDX-MS studies is that EX1 kinetics, while rare in most globular proteins, have been encountered quite frequently with membrane proteins.…”

Lysenin Channels as Sensors for Ions and Molecules

“…Among those biological tools, lysenin channels are attractive candidates for sensor development owing to their commercial availability, facile reconstitution into artificial membranes, extended stability, intrinsic regulatory mechanisms, and a large unobstructed opening. Lysenin is a 297-amino-acid pore-forming toxin extracted from the coelomic fluid of the earthworm E. fetida, which specifically interacts with sphingomyelin and oligomerizes into large conductance channels in artificial and natural lipid membrane systems [37][38][39][40][41][42][43][44][45][46]. Structural data achieved by employing X-ray crystallography, cryo-EM, and AFM indicate the existence of a large nonameric β-barrel pore (9-11 nm long, and 2-3 nm diameter) and no visible constrictions in the lumen [45][46][47][48][49][50][51].…”

Conserved cysteine dioxidation enhances membrane interaction of human Cl − intracellular channel 5

“…Our results suggest that CLICs may undergo membrane insertion following a mechanism reminiscent of that described for pore‐forming toxins, such as lysenin 57 . Secreted as a soluble monomer, this member of the aerolysin family exists as a monomer in solution.…”

“…Our results suggest that CLICs may undergo membrane insertion following a mechanism reminiscent of that described for pore-forming toxins, such as lysenin. 57 Secreted as a soluble monomer, this member of the aerolysin family exists as a monomer in solution. Upon exposure to sphingomyelin (SM)-containing membranes, lysenin undergoes a sequence of structural transitions, from water-soluble monomers into a membrane-embedded lytic β-barrel pore.…”